Hairpin-forming oligonucleotide hybridization probes with interactive label pairs, particularly fluorescent label pairs and fluorescer-quencher label pairs, are known. Tyagi et al., PCT application No. WO95/13399; Tyagi et al., PCT application No. WO97/39008; Tyagi and Kramer (1996) Nature Biotechnology 14:303. Preferred embodiments of these probes, labeled with a fluorophore and a quencher, are "dark", that is, have relatively little or no fluorescence, when free in solution but fluoresce when hybridized to their nucleic acid targets. We refer to such embodiments as "molecular beacon probes." They are constructed with a variety of fluorophores and are utilized in both end-point and real-time homogeneous assays, including multiplex assays. Tyagi et al. (1998) Nature Biotechnology 16:49; Kostrikis et al. (1998) Science 279:1228; Piatek et al. (1998) Nature Biotechnology 16:359. Hairpin-containing primers similarly labeled with a fluorophore and a quencher are also known. Nazarenko et al. (1997) Nucleic Acids Research 25:2516.
Fluorescence assay instruments that operate with a single stimulation wavelength are much less complicated and much less expensive than instruments that operate with multiple stimulation wavelengths. Almost all assay instruments, including sophisticated instruments costing many tens of thousands of dollars (U.S.) operate with single wavelength stimulation, even if the wavelength is selectable. Because every fluorophore has an optimal excitation wavelength, the choice of fluorophores to be used with a detection instrument having such a light source is limited. Certain fluorophores will be excited poorly or essentially not at all by the source. Red fluorophores such as tetramethylrhodamine (TMR) and Texas red are only minimally excited by a blue light source. A sophisticated, expensive instrument such as the Applied BioSystems 7700 PRISM, can detect fluorescence from TMR stimulated by a blue light source, but not Texas red. Less sophisticated, less expensive instruments can detect neither one. In multiplex assays for multiple targets using multiple hairpin probes or primers having differently colored fluorophores, it is desirable to be able to use four or even more fluorophores whose emission spectra have limited overlap, but the choice of fluorophores is limited by the use of single wavelength stimulation. Also, multiplex assays suffer from the drawback that the emission intensities of some of the fluorophores are very small both in absolute terms and also relative to other fluorophores in the assay. Furthermore, because the Stokes shift (the wavelength difference between the optimal excitation wavelength and optimal emission wavelength) of fluorophores is generally only a few nanometers, hairpin probes and primers whose fluorophores have emission maxima at or very close to the wavelength of the excitation source tend to suffer from background signal resulting from the source itself being detected by the fluorometer. This tends to be more pronounced when less expensive, unsophisticated detectors are employed.
An aspect of the present invention is probes and primers whose conformational change produces a detectable fluorescent signal having a greater Stokes shift than conventional hairpin probes and primers.
Another aspect of the present invention is a wider range of hairpin-forming probes and primers that are effectively quenched ("dark") in the absence of target but are better excited in the presence of target by a monochromatic light source.
Another aspect of the present invention is a group, or series, of hairpin-forming probes or primers containing differently colored fluorophores, all of which can be reasonably well excited by a single monochromatic light source.
Another aspect of the present invention is additional probes and primers suitable for use with single-wavelength stimulation instruments, including unsophisticated instruments for which the current choices are extremely limited.
Another aspect of the present invention is hairpin-forming probes and primers having reduced background signal as a consequence of the source itself not being detected.
Another aspect of the present invention is probes that fluoresce in one color if hybridized to target but change color if digested by a nuclease.
Additional aspects of the invention will be apparent from the description, including the claims, which follows.